CN111748581B - Method for transfecting dendritic cells by using hypoxia inducible factor recombinant expression plasmid - Google Patents

Method for transfecting dendritic cells by using hypoxia inducible factor recombinant expression plasmid Download PDF

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CN111748581B
CN111748581B CN201910898161.XA CN201910898161A CN111748581B CN 111748581 B CN111748581 B CN 111748581B CN 201910898161 A CN201910898161 A CN 201910898161A CN 111748581 B CN111748581 B CN 111748581B
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邓武
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Abstract

The invention provides a method for transfecting dendritic cells by using hypoxia inducible factor recombinant expression plasmids, and relates to the technical field of cell biology. The method provided by the invention comprises the following steps: mixing the dendritic cells, a liquid culture medium, the hypoxia inducible factor recombinant expression plasmid and a liposome solution, and standing to obtain a transfection system mixed solution dendritic cell culture solution; and mixing the transfection system mixed solution with a liquid culture medium and a cell factor to obtain a mixture, and culturing the mixture to obtain the transfected cells. By using the transfection method provided by the invention, the dendritic cells overexpress the Hif-1 alpha gene, so that the time required by maturation and activation of the dendritic cells can be obviously shortened, and the antigen presentation and T cell activation capabilities of the dendritic cells are improved.

Description

Method for transfecting dendritic cells by using hypoxia inducible factor recombinant expression plasmid
Technical Field
The invention relates to the technical field of cell biology, in particular to a method for transfecting dendritic cells by using hypoxia inducible factor recombinant expression plasmids.
Background
Tumors are one of the most serious chronic diseases that endanger human health. Clinically, the tumor is mainly treated by measures such as surgical excision, chemoradiotherapy, targeted therapy and the like, but most patients are easy to relapse and transfer after treatment. In recent years, with the development of medical and immunological techniques, immunotherapy has shown the potential to radically treat tumors, making it the most popular research direction in the current tumor therapy field.
Dendritic cells (DC cells) are currently known as the most prominent antigen-presenting cells. The DC cells are presented to immune effector cells through a self-surface histocompatibility complex I/II (MHCI/II) by taking up tumor cell surface related antigens, and meanwhile, the expression of a cell surface immune co-stimulatory molecule CD80/86 is remarkably increased, and the immune function of the DC cells is activated by combining with initial T cells; in addition, the activated DC cells can also jointly activate immunity by secreting cytokines such as IL-12, IFN-gamma and the like, thereby realizing the killing of tumor cells.
In recent years, with the intensive research on the immunity of tumors and tumors, the role of DC cells in immunotherapy has been developed. However, the existing method has the problems that the tumor cells usually proliferate rapidly, and take more nutrition and oxygen in the microenvironment, so that the microenvironment forms a phenomenon similar to hypoxia, so that peripheral immune cells can not acquire enough energy, and the growth and the function of DC cells are inhibited.
Disclosure of Invention
In view of the above, the present invention aims to provide a method for transfecting dendritic cells with a hypoxia inducible factor recombinant expression plasmid. By using the transfection method provided by the invention, the DC cell overexpresses the Hif-1 alpha gene, so that the time required by maturation and activation of the DC cell can be obviously shortened, and the antigen presenting and T cell activating capabilities of the DC cell are improved.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides a method for transfecting dendritic cells by using hypoxia inducible factor recombinant expression plasmids, which comprises the following steps: mixing the dendritic cells, a liquid culture medium, the hypoxia inducible factor recombinant expression plasmid and a liposome solution, and standing to obtain a transfection system mixed solution; mixing the transfection system mixed solution with a liquid culture medium and a cytokine to obtain a mixture, and culturing the mixture to obtain transfected cells;
the volume ratio of the number of the dendritic cells to the liquid culture medium is (0.8-1.2) x 1061mL of the total amount of the active ingredients;
the hypoxia inducible factor recombinant expression plasmid is pcDNA3-Hif-1 alpha P402A/P564A plasmid; the ratio of the mass of the pcDNA3-Hif-1 alpha P402A/P564A plasmid to the volume of the liquid culture medium is (500 ng-2 mu g) 1 mL;
the volume ratio of the liposome solution to the liquid culture medium is (2-3 mu L):1 mL.
Preferably, the liquid medium is a serum-free medium.
Preferably, the cytokines include granulocyte-macrophage colony stimulating factor and interleukin 4.
Preferably, the mass concentration of the granulocyte-macrophage colony stimulating factor in the mixture is 20-50 ng/mL.
Preferably, the mass concentration of the interleukin 4 in the mixture is 10-25 ng/mL.
Preferably, the mixed culture is cultured by using a carbon dioxide cell incubator.
Preferably, the time of the mixed culture is 6d to 8 d.
Preferably, the mixed culture is carried out until the 7d, lipopolysaccharide is further added, and the concentration of the lipopolysaccharide in the mixture is 100-200 ng/mL.
Preferably, the liposome comprises Lipofectamine2000 or TransLipidHL.
Preferably, the standing time is 15-35 min.
The invention provides a method for transfecting dendritic cells by using hypoxia inducible factor recombinant expression plasmids. By the transfection method, Dendritic Cells (DCs) are enabled to overexpress mutated Hif-1 alpha (P402A/P564A), and under the condition that the cells stably express the Hif-1 alpha, the maturation time of the DCs can be remarkably shortened, and the immune activation capacity of the DCs is improved.
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FIG. 1 is a diagram showing the results of in vitro culture of mouse DC cells, wherein A is the state of cell culture observed by a microscope; b, after the cells are cultured to the 7 th day and LPS is activated, detecting the expression level of the DC surface marker molecule CD-11c by flow cytometry; c, ELISA is used for detecting the expression level of the DC cell secretion cell factor IL-12;
FIG. 2 is a diagram showing RT-qPCR method for detecting the expression level of Hif-1a gene in transfected DC cells;
FIG. 3 is the detection of maturation, differentiation and secretion indexes of transfected DC cells; wherein A is the proliferation speed of DC cells after MTT method comparison transfection; b, detecting the expression level changes of DC specific molecules CD-11c and activation marks CD80/CD86 on the surface of the DC cells after transfection by flow cytometry; C-D is ELISA method for detecting the change of the secretion of immune activation cell factor IL-12 and IFN-gamma level of DC cells after transfection.
Detailed Description
The invention provides a method for transfecting dendritic cells by using hypoxia inducible factor recombinant expression plasmids, which comprises the following steps: mixing the dendritic cells, the liquid culture medium, the hypoxia inducible factor recombinant expression plasmid and the liposome, and standing to obtain a transfection system mixed solution; mixing the transfection system mixed solution with a liquid culture medium and a cytokine to obtain a mixture, and culturing the mixture to obtain transfected cells; the volume ratio of the number of the dendritic cells to the liquid culture medium is (0.8-1.2) x 1061mL of the total amount of the active ingredients; the hypoxia inducible factor recombinant expression plasmid is pcDNA3-Hif-1 alpha P402A/P564A plasmid; the ratio of the mass of the pcDNA3-Hif-1 alpha P402A/P564A plasmid to the volume of the liquid culture medium is (500 ng-2 mu g) 1 mL; the above-mentionedThe volume ratio of the liposome solution to the liquid culture medium is (2-3 μ L):1 mL.
In the present invention, the Dendritic Cells (DCs) are preferably mouse DC cells; the mouse DC cells are preferably extracted and separated by the following method comprising the following steps: killing 6-8 weeks old female mice and then disinfecting; taking femurs on both sides under a sterile state, shearing off two ends of a bone, and taking out bone marrow cells; filtering the residual tissue fragments, collecting cell suspension, and removing red blood cells; centrifuging the suspension, removing the supernatant, resuspending with serum-free cell culture medium, counting cells under microscope at 1 × 105～5×105Individual cell/cm2Inoculating to cell culture dish, and standing CO2Culturing the cells in an incubator, and adding cytokines to induce bone marrow cells to differentiate into DC cells; changing the cell culture solution for half amount every 2d, and supplementing the above cell factor according to the original concentration; cells were cultured to 7d and Lipopolysaccharide (LPS) was added to stimulate maturation of DC cells.
In the present invention, the disinfection is preferably performed by using a 75% ethanol solution, and the disinfection time is preferably 5min to 15min, and more preferably 10 min.
In the present invention, the filtration of the residual tissue fragments is preferably performed using a screen of 180 to 220 mesh, more preferably 200 mesh.
In the invention, the erythrocyte is preferably removed by adopting a method of removing the erythrocyte by using erythrocyte lysate.
In the present invention, the cell culture medium is preferably X-VIVO-15 serum-free medium. The X-VIVO-15 serum-free culture medium is a commercial product, is produced by the company LONZA in Switzerland, and is specially used for culturing immune cells, and the types of the X-VIVO series serum-free culture medium are 10, 15, 20 and the like. The product model used in the embodiment of the invention is X-VIVO-15.
In the present invention, the CO is2The time for the cell incubator culture is preferably 5min to 15min, more preferably 10 min.
In the invention, the centrifugal speed is preferably 700rpm to 900rpm, more preferably 800 rpm; the centrifugation time is preferably 4 to 6min, more preferably 5 min.
In the method, in the extraction and separation of DC cells, the cytokines are preferably granulocyte-macrophage colony stimulating factor (GM-CSF) and interleukin 4 (IL-4); the mass concentration of the GM-CSF is preferably 20-50 ng/mL, and more preferably 30-40 ng/mL; the mass concentration of the IL-4 is preferably 10-25 ng/mL, and more preferably 15-20 ng/mL.
In the transfection method of the present invention, the ratio of the number of dendritic cells to the volume of the liquid medium is preferably (0.8 to 1.2). times.106 cells/1 mL, and more preferably 1X 1061mL of the total amount of the active ingredients; the liquid culture medium is preferably a serum-free culture medium, and the liquid culture medium is RPMI-1640 cell culture medium (Sigma-aldrich company) without adding serum components; the main components comprise: 0.1g/L of calcium nitrate tetrahydrate; 0.04884g/L magnesium sulfate (anhydrous); 0.4g/L of potassium chloride; 2g/L of sodium bicarbonate; 6g/L of sodium chloride; 0.8g/L disodium hydrogen phosphate (anhydrous); 0.2g/L of L-arginine; 0.05g/L of L-asparagine; 0.02g/L of L-aspartic acid; 0.0652g/L of L-cystine dihydrochloride; 0.02g/L of L-glutamic acid; 0.3g/L of L-glutamine; 0.01g/L of glycine; l-histidine 0.015 g/L; 0.02g/L of hydroxy-L-proline; 0.05g/L of L-isoleucine; l-leucine 0.05 g/L; l-lysine hydrochloride 0.04 g/L; l-methionine 0.015 g/L; 0.015g/L of L-phenylpropyl alanine; l-proline 0.02 g/L; l-serine 0.03 g/L; 0.02g/L of L-threonine; l-tryptophan 0.005 g/L; 0.02883g/L of L-tyrosine disodium dihydrate; 0.02g/L of L-valine, trace vitamins and the like. In the present embodiment, X-VIVO-15 serum-free medium is preferable.
The hypoxia inducible factor recombinant expression plasmid is pcDNA3-Hif-1 alpha P402A/P564A plasmid; the ratio of the mass of the pcDNA3-Hif-1 alpha P402A/P564A plasmid to the volume of the liquid culture medium is (500 ng-2 mu g):1mL, preferably (800-1500 ng):1mL, and more preferably 1000ng:1 mL.
The volume ratio of the liposome solution to the liquid culture medium is (2-3 muL): 1mL, preferably 2.5 muL: 1 mL. In the present invention, the liposome is preferably Lipofectamine2000 or TransLipidHL, and the liposome may be prepared from a conventional commercially available product. In the invention, the Lipofectamine2000, abbreviated as "Lipo 2000", is a product of Thermofeisher company, and the Lipofectamine2000 is prepared by sucking a specific volume of liposome and adding the liposome into a proper volume of cell culture solution to mix uniformly during specific operation.
In the transfection method of the present invention, the cytokine preferably comprises granulocyte-macrophage colony stimulating factor and interleukin 4, and the mass concentration of the granulocyte-macrophage colony stimulating factor in the mixture is preferably 20-50 ng/mL, and more preferably 30-40 ng/mL; the mass concentration of the interleukin 4 in the mixture is preferably 10-25 ng/mL, and more preferably 15-20 ng/mL.
In the invention, the mixed culture is carried out by adopting a carbon dioxide cell incubator, the culture temperature is 37 ℃, and the volume concentration of carbon dioxide is 5%. In the mixed culture of the invention to the 7 th day, the method also preferably comprises adding lipopolysaccharide, and the concentration of the lipopolysaccharide in the mixture is preferably 100-200 ng/mL, and more preferably 130-160 ng/mL. In the present invention, the addition of lipopolysaccharide has the effect of stimulating the maturation of DC cells.
In the transfection method of the present invention, the time for culturing is preferably 6d to 8d, more preferably 7 d.
The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. It is to be understood that the described embodiments are merely exemplary of the invention, and not restrictive of the full scope of the invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example 1
1. Extraction and isolation of mouse DC cells
Taking a female C57 strain mouse with the age of 6-8 weeks, taking off the neck, and after killing, soaking and disinfecting for 10min by using 75% alcohol; taking out the femurs on both sides in a sterile state; the bone ends were cut off, and the bone marrow cells were washed out by filling sterile 1 XPBS buffer using a 1ml disposable syringe, inserting into the bone marrow cavity and washing 3 times. Filtering the residual tissue debris with a 200 mesh screen, collecting the cell suspension, adding 5 Xerythrocyte lysate, 5% CO at 37 deg.C2Reacting in a cell incubator for 10min, and cracking red blood cells in the suspension; the suspension is centrifuged and the suspension is centrifuged,800rpm/min, 5min, discarding the supernatant, resuspending with serum-free cell culture medium (X-VIVO-15 serum-free medium), counting cells under microscope at 1 × 105Individual cell/cm2Inoculating to cell culture dish, standing at 37 deg.C and 5% CO2Culturing in incubator, adding 20ng/ml GM-CSF and 10ng/ml IL-4 to induce differentiation of bone marrow cells to DC cells. Cell status, morphology and photographs were observed daily. Half of the cell culture solution is changed every 2 days, and the cell factors are supplemented according to the original concentration. Cells were cultured to 7d and maturation of DC cells was stimulated by addition of 100ng/ml Lipopolysaccharide (LPS) (FIG. 1).
2. Identification of DC cell purity and Activity
Collecting sufficient cells before adding LPS into the cell culture medium on the 6 th day of culture and after stimulating for 24h, and detecting the positive proportion of CD11c and the expression activation level of CD80/CD86 of the cells by flow cytometry; collecting cell culture supernatant, and determining the levels of cell secreted immune activation cytokines IL-12 and IFN-gamma by enzyme-linked immunosorbent assay (ELISA).
3. Transfection of Hif-1 alpha (P402A/P564A) Gene into DC cells
And (3) after the DC cells obtained in the step (1) are cultured for 24 hours, observing the cell state, and performing gene transfection operation when the growth is good. 1X 10 per culture dish6Calculating each cell, taking 2 sterile EP tubes, respectively adding 500 μ l of serum-free culture medium, adding 2 μ g of purified pcDNA3-Hif-1 α P402A/P564A plasmid into 1 tube, adding 2 μ l of liposome (Lipo2000) into the other 1 tube, respectively shaking gently, mixing, and standing for 5 min; then, the two tubes were mixed, gently shaken and mixed, and left to stand for 20 min.
Then gently sucking out cell culture solution, rinsing with sterile 1 × PBS buffer solution for 1 time, adding the culture solution and cytokine of step 1 again, gently shaking and mixing, placing the culture dish back at 37 deg.C and 5% CO2And continuously culturing the cell incubator. Observing the growth state of the cells every day, and continuously updating the cell culture solution according to the requirements of the step 1.
4. Characterization of transfection efficiency
A part of cells were collected on days 1, 4 and 7 after cell transfection, total cellular RNA was extracted, and the expression level of Hif-1. alpha. in the cells was measured by a real-time quantitative PCR (RT-qPCR) method (FIG. 2).
5. Detection of maturation, differentiation and secretion indexes of transfected DC cells
Inoculating cells in a 96-well plate, detecting by adopting an MTT colorimetric method on days 1, 3 and 5 after cell transfection, and comparing the change of the proliferation capacity of the transfected DC cells;
collecting partial cells on 5 th day after cell transfection, and detecting the expression activation level of CD11c and CD80/CD86 of the cells by flow cytometry; cell culture supernatants were collected on day 7 of culture and assayed for levels of IL-12, IFN-. gamma.which are cell-secreted immune-activating cytokines by enzyme-linked immunosorbent assay (ELISA) (FIG. 3).
Example 2
1. Extraction and isolation of mouse DC cells
Taking a female C57 strain mouse with the age of 6-8 weeks, taking off the neck, and after killing, soaking and disinfecting for 10min by using 75% alcohol; taking the bilateral femur and tibia under aseptic condition); the bone ends were cut off, and the bone marrow cells were washed out by filling sterile 1 XPBS buffer using a 1ml disposable syringe, inserting into the bone marrow cavity and washing 4 times. Filtering the residual tissue debris with a 200 mesh screen, collecting the cell suspension, adding 5 Xerythrocyte lysate, 5% CO at 37 deg.C2Reacting in a cell incubator for 10min, and cracking red blood cells in the suspension; centrifuging the suspension, removing supernatant at 800rpm/min for 5min, resuspending with serum-free cell culture medium (X-VIVO-15 serum-free medium), counting cells under microscope, and measuring by 5 × 105Individual cell/cm2Inoculating to cell culture dish, standing at 37 deg.C and 5% CO2Culturing in incubator, adding GM-CSF 50ng/ml and IL-4 25ng/ml to induce differentiation of bone marrow cells to DC cells. Cell status, morphology and photographs were observed daily. Half of the cell culture solution is changed every 2 days, and the cell factors are supplemented according to the original concentration. Cells were cultured to 7d and maturation of DC cells was stimulated by addition of 200ng/ml Lipopolysaccharide (LPS).
2. Identification of DC cell purity and Activity
Collecting sufficient cells before adding LPS into the cell culture medium on the 6 th day of culture and after stimulating for 24h, and detecting the positive proportion of CD11c and the expression activation level of CD80/CD86 of the cells by flow cytometry; collecting cell culture supernatant, and determining the levels of cell secreted immune activation cytokines IL-12 and IFN-gamma by enzyme-linked immunosorbent assay (ELISA).
3. Transfection of Hif-1 alpha (P402A/P564A) Gene into DC cells
And (3) after the DC cells obtained in the step (1) are cultured for 24 hours, observing the cell state, and performing gene transfection operation when the growth is good. 1X 10 per culture dish6Calculating each cell, taking 2 sterile EP tubes, respectively adding 500 μ l of serum-free culture medium, adding 2 μ g of purified pcDNA3-Hif-1 α P402A/P564A plasmid into 1 tube, adding 2 μ l of liposome (Lipo2000) into the other 1 tube, respectively shaking gently, mixing, and standing for 5 min; then, the two tubes were mixed, gently shaken and mixed, and left to stand for 20 min.
Then gently sucking out cell culture solution, rinsing with sterile 1 × PBS buffer solution for 1 time, adding the culture solution and cytokine of step 1 again, gently shaking and mixing, placing the culture dish back at 37 deg.C and 5% CO2And continuously culturing the cell incubator. Observing the growth state of the cells every day, and continuously updating the cell culture solution according to the requirements of the step 1.
4. Characterization of transfection efficiency
Collecting partial cells on days 1, 4 and 7 after cell transfection, extracting total RNA of the cells, and determining the expression level of Hif-1 alpha of the cells by a real-time quantitative PCR (RT-qPCR) method.
5. Detection of maturation, differentiation and secretion indexes of transfected DC cells
Inoculating cells in a 96-well plate, detecting by adopting an MTT colorimetric method on days 1, 3 and 5 after cell transfection, and comparing the change of the proliferation capacity of the transfected DC cells;
collecting partial cells on days 1, 3 and 5 after cell transfection, and detecting the expression activation level of CD11c and CD80/CD86 of the cells by flow cytometry;
collecting cell culture supernatant, and determining the levels of cell secreted immune activation cytokines IL-12 and IFN-gamma by enzyme-linked immunosorbent assay (ELISA).
Example 3
Extraction and isolation of mouse DC cells
Taking a female C57 strain mouse with the age of 6-8 weeks, taking off the neck, and after killing, soaking and disinfecting for 10min by using 75% alcohol; taking out the femurs on both sides in a sterile state; the bone ends were cut off, and the bone marrow cells were washed out by filling sterile 1 XPBS buffer using a 1ml disposable syringe, inserting into the bone marrow cavity and washing 4 times. Filtering the residual tissue debris with a 200 mesh screen, collecting the cell suspension, adding 5 Xerythrocyte lysate, 5% CO at 37 deg.C2Reacting in a cell incubator for 10min, and cracking red blood cells in the suspension; centrifuging the suspension, removing supernatant at 800rpm/min for 5min, resuspending with serum-free cell culture medium (X-VIVO-15 serum-free medium), counting cells under microscope, and performing 2 × 10 cell counting5Individual cell/cm2Inoculating to cell culture dish, standing at 37 deg.C and 5% CO2Culturing in incubator, adding GM-CSF 40ng/ml and IL-4 20ng/ml to induce differentiation of bone marrow cells to DC cells. Cell status, morphology and photographs were observed daily. Half of the cell culture solution is changed every 2 days, and the cell factors are supplemented according to the original concentration. Cells were cultured to 7d and maturation of DC cells was stimulated by addition of 200ng/ml Lipopolysaccharide (LPS).
2. Identification of DC cell purity and Activity
Collecting sufficient cells before adding LPS into the cell culture medium on the 6 th day of culture and after stimulating for 24h, and detecting the positive proportion of CD11c and the expression activation level of CD80/CD86 of the cells by flow cytometry; collecting cell culture supernatant, and determining the levels of cell secreted immune activation cytokines IL-12 and IFN-gamma by enzyme-linked immunosorbent assay (ELISA).
3. Transfection of Hif-1 alpha (P402A/P564A) Gene into DC cells
And (3) after the DC cells obtained in the step (1) are cultured for 24 hours, observing the cell state, and performing gene transfection operation when the growth is good. 1X 10 per culture dish6Calculating each cell, taking 2 sterile EP tubes, respectively adding 500 μ l of serum-free culture medium, adding 2 μ g of purified pcDNA3-Hif-1 α P402A/P564A plasmid into 1 tube, adding 2 μ l of liposome (Lipo2000) into the other 1 tube, respectively shaking gently, mixing, and standing for 5 min; then, the two tubes were mixed, gently shaken and mixed, and left to stand for 20 min.
Then the cell culture solution is gently sucked out, and the sterile 1 functionRinsing with PBS buffer solution for 1 time, adding the culture solution and cytokine from step 1, shaking gently, mixing, placing the culture dish at 37 deg.C and 5% CO2And continuously culturing the cell incubator. Observing the growth state of the cells every day, and continuously updating the cell culture solution according to the requirements of the step 1.
4. Characterization of transfection efficiency
Collecting partial cells on days 1, 4 and 7 after cell transfection, extracting total RNA of the cells, and determining the expression level of Hif-1 alpha of the cells by a real-time quantitative PCR (RT-qPCR) method.
5. Detection of maturation, differentiation and secretion indexes of transfected DC cells
Inoculating cells in a 96-well plate, detecting by adopting an MTT colorimetric method on days 1, 3 and 5 after cell transfection, and comparing the change of the proliferation capacity of the transfected DC cells;
collecting partial cells on days 1, 3 and 5 after cell transfection, and detecting the expression activation level of CD11c and CD80/CD86 of the cells by flow cytometry;
collecting cell culture supernatant, and determining the levels of cell secreted immune activation cytokines IL-12 and IFN-gamma by enzyme-linked immunosorbent assay (ELISA).
From the above results, it was found that the proliferation rate of DC cells transfected with Hif-1 α (P402A/P564A) was high, and the expression purity of CD11c, a characteristic marker of DC cells, was superior to that of the conventional culture method. After immune activation, the expression level of the co-stimulatory factor CD80/86 and the secretion level of the immune activation cytokine are both higher than those of the untransfected DC cells. Has obvious technical effect.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.

Claims (9)

1. A method for transfecting dendritic cells by using a hypoxia inducible factor recombinant expression plasmid is characterized by comprising the following steps: mixing the dendritic cells, a liquid culture medium, the hypoxia inducible factor recombinant expression plasmid and a liposome solution, and standing to obtain a transfection system mixed solution; mixing the transfection system mixed solution with a liquid culture medium and a cytokine to obtain a mixture, and culturing the mixture to obtain transfected cells;
the volume ratio of the number of the dendritic cells to the liquid culture medium is (0.8-1.2) x 1061mL of the total amount of the active ingredients;
the hypoxia inducible factor recombinant expression plasmid is pcDNA3-Hif-1 alpha P402A/P564A plasmid; the ratio of the mass of the pcDNA3-Hif-1 alpha P402A/P564A plasmid to the volume of the liquid culture medium is (500 ng-2 mu g) 1 mL;
the volume ratio of the liposome solution to the liquid culture medium is (2-3 mu L) to 1 mL;
the liposome comprises Lipofectamine2000 or TransLipid HL.
2. The method of claim 1, wherein the liquid medium is a serum-free medium.
3. The method of claim 1, wherein the cytokines comprise granulocyte-macrophage colony stimulating factor and interleukin-4.
4. The method according to claim 3, wherein the mass concentration of granulocyte-macrophage colony stimulating factor in the mixture is 20-50 ng/mL.
5. The method according to claim 3, wherein the mass concentration of interleukin 4 in the mixture is 10-25 ng/mL.
6. The method of claim 1, wherein the mixture is cultured using a carbon dioxide cell incubator.
7. The method of claim 1, wherein the mixture is incubated for a period of time ranging from 6 days to 8 days.
8. The method according to claim 1 or 7, wherein the mixture is cultured to 7d, and further comprises adding lipopolysaccharide, and the concentration of the lipopolysaccharide in the mixture is 100-200 ng/mL.
9. The method according to claim 1, wherein the standing time is 15-35 min.
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